Capillary LC-MS2 at the attomole level for monitoring and discovering endogenous peptides in microdialysis samples collected in vivo

Fused-silica capillary LC columns (25-mum i.d.) with 3-mum-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated for high-sensitivity LC-MS2. Column preparation involved co nstructing frits by in situ photopolymerization of glycidyl methacrylate an d trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the column outlet to a fine tip with a CO2 laser puller, and slurr y-packing the column with 5-mum reversed-phase particles. Large-volume inje ctions were facilitated by an automated two-pump system that allowed high-f low rates for sample loading and low-flow rates for elution. Small electros pray emitters, low elution flow rates, and optimization of gradient steepne ss allowed a detection limit of 4 amol, corresponding to 2 pM for 1.8 muL i njected on-column, for a mixture of peptides dissolved in artificial cerebr al spinal fluid. The system was coupled on-line to microdialysis sampling a nd was used to monitor and discover endogenous neuropeptides from the globu s pallidus of anesthetized. male Sprague-Dawley rats. Time-segmented MS2 sc ans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephal in were 60 +/- 30 and 70 +/- 20 pM while K-(+)-stimulated levels were 1900 +/- 500 and 1300 +/- 300 pM, respectively (n = 7). Data-dependent and time- segmented MS2 scans revealed several unknown peptides that were present in dialysate. One of the unknowns was identified as peptide I1-10 (SPQLEDEAKE) , a novel product of preproenkephalin A processing, using MS2, MS3, and dat abase searching.